Cholesterol Reduces Pardaxin's Dynamics—A Barrel-Stave Mechanism of Membrane Disruption Investigated by Solid-State NMR

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1798 (2010) 223–227 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Cholesterol reduces pardaxin's dynamics—a barrel-stave mechanism of membrane disruption investigated by solid-state NMR Ayyalusamy Ramamoorthy ⁎, Dong-Kuk Lee 1, Tennaru Narasimhaswamy 2, Ravi P.R. Nanga Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA article info abstract Article history: While high-resolution 3D structures reveal the locations of all atoms in a molecule, it is the dynamics that Received 9 June 2009 correlates the structure with the function of a biological molecule. The complete characterization of Received in revised form 11 August 2009 dynamics of a membrane protein is in general complex. In this study, we report the influence of dynamics on Accepted 15 August 2009 the channel-forming function of pardaxin using chemical shifts and dipolar couplings measured from 2D Available online 28 August 2009 broadband-PISEMA experiments on mechanically aligned phospholipids bilayers. Pardaxin is a 33-residue antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus, which Keywords: Dynamic functions via either a carpet-type or barrel-stave mechanism depending on the membrane composition. Our Pardaxin results reveal that the presence of cholesterol significantly reduces the backbone motion and the tilt angle of Membrane orientation the C-terminal amphipathic helix of pardaxin. In addition, a correlation between the dynamics-induced Barrel-stave heterogeneity in the tilt of the C-terminal helix and the membrane disrupting activity of pardaxin by the Antimicrobial peptide barrel-stave mechanism is established. This correlation is in excellent agreement with the absence of hemolytic activity for the derivatives of pardaxin. These results explain the role of cholesterol in the selectivity of the broad-spectrum of antimicrobial activities of pardaxin. © 2009 Elsevier B.V. All rights reserved. 1. Introduction significant need for biophysical studies to identify the roles of membrane components on the function of AMPs. In this study, we Design of novel antibiotic compounds to overcome the increasing report atomic-level insights into the role of cholesterol on the bacterial resistance towards conventional antibiotics is of consider- function of pardaxin using solid-state NMR experiments on mechan- able current importance [1]. Numerous studies have reported the ically aligned model phospholipid membranes. Cholesterol is abun- isolation, characterization, biological functions, and potential phar- dantly present in mammalian membranes, but not in bacterial maceutical applications of naturally occurring AMPs [2,3]. A variety of membranes, and has been thought to be crucial in reducing the biochemical and biophysical approaches have reported mechanisms toxicity of AMPs. of membrane permeation/disruption, bioavailability, and synergistic Pardaxin is a 33-residue antimicrobial peptide originally isolated activities of AMPs [4–7]. High-resolution cutting-edge techniques from the secretions from the Red Sea Moses sole, Pardachirus have been used to obtain atomic-level insights into the structure, marmoratus. Pardaxin has been shown to exhibit a broad-spectrum dynamics, folding, oligomerization, and membrane orientation of of antimicrobial activities at very low concentrations, to act as a AMPs and their interactions with lipid membrane [8]. These studies channel-forming neurotoxin, and also to cause cell death by necrosis have been utilized in the design of potent peptide antibiotics [9–11]. at slightly higher concentrations [16–21]. Amino acid sequences of In spite of these valuable studies, unfortunately, only a very few pardaxin peptides are given in Fig. 1 along with the 3D NMR structure peptides have been found to be suitable for pharmaceutical appli- of the peptide. An early NMR study on pardaxin in a TFE:water cations [5,12–15]. One of the major difficulties in designing potent solution reported a helix–hinge–helix structure similar to that of AMPs is determining the role of membrane components that play melittin [22]. But a recent NMR study reported the more biologically important roles in the selectivity of AMPs. Therefore, there is a relevant structure of the peptide in a membrane environment, where the structure of the N-terminal domain significantly differs from that obtained from TFE:water structure (Fig. 1) [23]. Recent NMR studies have also reported the membrane orientation, role of membrane ⁎ Corresponding author. Tel.: +1 734 647 6572. composition, and potential mechanisms of the membrane permeation E-mail address: [email protected] (A. Ramamoorthy). of pardaxin [23,24]. The antimicrobial activities of peptide fragments 1 Present address: Department of Fine Chemistry, Seoul National University of corresponding to 1–11, 1–18, 1–22, 10–33, and 12–33 residues of Technology, Seoul, Korea. 2 Present Address: Polymer lab, Central Leather Research Institute, Adayaru, Chennai pardaxin and several mutants have also been reported. The amidated 600 020, Tamil Nadu, India. forms of the 22-residues C-terminal fragment and the 11 amino acid 0005-2736/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2009.08.012 224 A. Ramamoorthy et al. / Biochimica et Biophysica Acta 1798 (2010) 223–227 2. Materials and methods 2.1. Materials Methanol and chloroform were purchased from Aldrich Chemical Inc. (Milwaukee, WI). Phospholipids, cholesterol, and naphthalene were purchased from Avanti Polar Lipids (Alabaster, AL), Sigma (St. Louis, MO), and Fisher Scientific (Pittsburg, PA), respectively. All these chemicals were used without further purification. All peptides were synthesized and purified as explained in our earlier publication [24]. Mechanically aligned bilayer samples were prepared using the naphthalene procedure as explained in our previous publications [29]. 2.2. Solid-state NMR All of the solid-state NMR experiments were performed on a Chemagnetics/Varian Infinity 400 MHz spectrometer operating at resonance frequencies of 400.138, 161.979, and 40.55 MHz for 1H, 31P, and 15N nuclei, respectively using home-built flat-coil probes. 31P chemical shift spectra obtained using a spin-echo sequence (90°–τ– 180°–τ-acquisition; τ=70 μs) with a 90° pulse length of 3.5 μs under 40 kHz proton decoupling were used to confirm the alignment of each mechanically aligned bilayer samples after an equilibration for about 30 min at a chosen temperature before recording the spectrum. All reported solid-state NMR experiments were performed with the bilayer normal of the sample oriented parallel to the external magnetic field. After optimization of the experimental conditions for sensitivity, resolution, and sample stability using a series of 1D NMR experiments as explained elsewhere [28], 2D BB-PISEMA experiments were performed as explained in our previous publication [26]. A ramp cross-polarization sequence with a 1H π/2 pulse length of 3 μs, 50 kHz cross-polarization power, a 1H decoupling of 75 kHz during acquisition, and a 2 s recycle delay were used in 1D experiments. An RF field strength of 62.5 kHz was used for the Lee–Goldburg off-resonance pulses during the t1 period of BB-PISEMA. 32 t1 increments each with 3600 scans were recorded for 2D BB-PISEMA spectra. All experimental data were processed using the Spinsight (Chemagnetics/Varian) software on a Sun SPARC workstation. 3. Results A previous solid-state NMR study reported a transmembrane orientation for the C-terminal helix of pardaxin in DMPC and a membrane surface orientation in POPC bilayers [24]. Since we are Fig. 1. Pardaxin—a cationic antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus. (A) Amino acid sequences of pardaxin peptides. interested in probing the role of cholesterol on the dynamics of the Pa4 was used in this study. (B) NMR structure of pardaxin determined from DPC peptide and its implications for the barrel-stave mechanism of micelles [23]. (C) A helical wheel representation of the C-terminal amphipathic domain membrane disruption by pardaxin, we chose to use DMPC and of pardaxin. DMPG lipids for this study in which the peptide is in a transmembrane orientation. Two types of model membrane compositions were used in this study: 3:1 DMPC:DMPG bilayers and DMPC bilayers containing N-terminal domain exhibit antimicrobial activity only against Gram- 15 mol% cholesterol to understand the role of cholesterol present in positive bacteria 17], whereas the fragment 1–18 has been shown to mammalian membranes. 2D BB-PISEMA spectra of these samples at be active against Escherichia coli but not against Gram-positive various temperatures are given in Fig. 2. A single peak observed in the bacteria [25]. These differences in the activities of the derivatives of 15N chemical shift region of 175 to 183 ppm and a 15N–1H dipolar pardaxin are not well understood. coupling frequency of 8–9.3 kHz confirms the transmembrane In this study, we show that the presence of cholesterol reduces the orientation of the C-terminal helix of pardaxin under this condition. dynamics of pardaxin but does not influence the membrane 2D experiments were performed to measure the influence of orientation of pardaxin. Influences of temperature and cholesterol temperature in the presence

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